[Histonet] Comments on BRDU detection

[andres]

Dear "Histonetters",

I've just gone over all the archived messages in the list about "BRDU" and 
I have to say I've learnt a lot about it (I confirm the utility of the 
list, and congratulate you all for make it work) . However, I still have a 
question and a comment I wanted to post to you.

We are doing fluorescent BrdU detection on 20 um crystotat sections of 
chick embryos (parafolmadehyde fixed and sucrose cryoprotected). First, I'd 
like to share my results with you as it might may be of any help, second 
ask if someone has seen the same kind of thing, and finally, try to find a 
way to solve the following:

When we treat the sections with 2N HCl for 30 min RT, appart from the 
poorly conserved nuclear morphology that seem to be common for many 
colleagues (although we still get DAPI staining reasonably conserved), we 
ONLY get BrdU staining in the 5-15 um surface of the section (as evaluated 
by confocal z-scanning). This is specific for BrdU detection, since double 
immunofluorescence with other antibodies penetrate the whole 20 um of the 
specimen. These low penetration results are very consistant, and it's not a 
problem of particular solutions or tissue sections.

Increasing the time and temperature of the acid treatment (e.g. 2N HCl for 
2 hs, RT or 37ºC) seems to improve the "penetration" of the staining, 
although DAPI or TOPRO staining is lost. More interestingly,  the 
improvement is only seen in the mesenchymal tissues, but NOT in more dense 
tissues (particularly, the neuroepithelium of the brain or spinal cord). So 
in those tissues we still get ONLY about 5 um-deep staining. I suspect this 
is related to insufficient DNA denaturation in those tissues, but as I said 
this is already quite a strong treatment.

Most of the people use the HCl treatment, even with shorter times, but I 
haven't seen any report of poor penetration of the BrdU stain, so obviously 
I wonder if there's anything we're doing wrong. However, we also think that 
for many people this issue may not be a problem,  and may not even realized 
about it, specially if you use chromogenic reactions like DAB. 
UNfortunately, for some purposes it may actually be very important, so I'd 
like to know if any of you have also checked and/or seen this effect, 
specially those of you working with tight epithelia or nervous system.

Also, if there's any alternative to overcome the acid treatment (we've 
tried citric acid Antigen retrieval but didn't get any signal at all, and 
formamide treatment doesn't improve it either).

I apologize for the long message, and I hope to get your inputs or comments 
on this.

Once again thank you,

A


Andrés Kamaid
Developmental Biology Group
Universitat Pompeu Fabra
Barcelona -Spain